Synergy-Based Biocontrol of Plant Pathogens

ABSTRACT

Methods and compositions are provided for control of pathogenic fungal or oomycetous infection.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/287,607 filed Oct. 6, 2016, which is a Continuation of U.S.application Ser. No. 14/537,620, filed Nov. 10, 2014, now U.S. Pat. No.9,485,994, which claims priority to U.S. Provisional Application61/902,046, filed Nov. 8, 2013, the contents of which are herebyincorporated in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Fungal and oomycetous pathogens can cause significant damage to a widevariety of commercially important plant varieties including crops andornamental plants. Such pathogens can kill plants, reduce yield, reduceplant strength (e.g., decrease resistance to lodging), cause symptoms ofmineral deficiency, and predispose plants to infection by otherpathogens. As such, there is great interest in developing compositionsand methods for control of fungal and oomycetous pathogens.

Fungal and oomycetous pathogens are typically controlled by use ofsynthetic chemicals (e.g., fungicides). However, effective syntheticchemicals can be dangerous, toxic, and expensive. In some countries,certain fungicides or anti-oomycetous chemicals have been restricted orbanned for these and other reasons. Therefore, there is great interestin developing fungal control methods and compositions that do not relyon synthetic chemical fungicides or anti-oomycetous compounds, or reducethe use of such chemicals.

Biocontrol agents are a promising candidate for reducing or eliminatingthe need for such chemicals. Biocontrol agents are typicallymicroorganisms, such as bacteria, or one or more products thereof, thatare applied to a plant or a plant propagation material (e.g., soil) tocontrol a pathogen. However, there are a limited number of commerciallyavailable biocontrol agents. For these and other reasons, there remainsa need to further develop methods and compositions containing biocontrolagents for the control of pathogenic fungi and Oomycetes.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method ofcultivating a plant resistant to a fungal or oomycetous infectioncomprising the steps of: a) contacting said plant with bacteria of thegenus Collimonas, or a product thereof; and b) contacting said plantwith bacteria of the genus Bacillus, or a product thereof, wherein saidplant shows a greater resistance to the infection or exhibits reducedsymptoms of fungal or oomycetous infection relative to a non-contactedplant. In some embodiments, the plant is cultivated in soil or otherplanting media that does not otherwise contain bacteria of the genusCollimonas. In some embodiments, said plant is a plant selected from thegroup consisting of tomato, potato, sweet potato, cassava, beets,ginger, horseradish, radish, ginseng, turnip, any root or tuber crop,pepper, eggplant, ground cherry, tomatillo, okra, other fruitingvegetables, cucumber cantaloupe, melon, muskmelon, squash, watermelonand other cucurbit plants.

In some embodiments, the part of said plant contacted with bacteria ofthe genus Collimonas or a product thereof is a root of said plant. Insome embodiments, the part of said plant contacted with bacteria of thegenus Bacillus or a product thereof is a root of said plant. In someembodiments, the part of said plant contacted with bacteria of the genusCollimonas or a product thereof is the seed of said plant. In someembodiments, the part of said plant contacted with bacteria of the genusBacillus or a product thereof is the seed of said plant. In someembodiments, the part of said plant contacted with bacteria of the genusCollimonas or a product thereof is the foliage of said plant. In someembodiments, the part of said plant contacted with bacteria of the genusBacillus or a product thereof is the foliage of said plant.

In some embodiments, the contacting of a) comprises contacting the plantwith bacteria of the genus Collimonas and a product thereof. In someembodiments, the contacting of b) comprises contacting the plant withbacteria of the genus Bacillus and a product thereof. In someembodiments, the contacting of a) comprises contacting the plant withbacteria of the genus Collimonas and a product thereof, and thecontacting of b) comprises contacting the plant with bacteria of thegenus Bacillus and a product thereof.

In some embodiments, the fungal or oomycetous infection comprises aninfection by a fungal pathogen from a class selected from the groupconsisting of Plasmodiophoromycetes, Oomycetes, Chytridiomycetes,Zygomycetes, Ascomycetes, Basidiomycetes, Deuteromycetes,Sordariomycetes, and combinations thereof. In some cases, the fungal oroomycetous pathogen is from the class Oomycetes. In some embodiments,said fungal or oomycetous infection comprises infection by a fungalpathogen from a genus selected from the group consisting of Fusarium,Geotrichum, Aspergillus, Alternaria, Botryosphaeria, Colletotrichum,Magnaporthe, Verticillium, Cryphonectria, Botrytis, Monilinia,Sclerotium, Rhizoctonia, and combinations thereof. In some embodiments,said fungal or oomycetous infection comprises infection by an Oomycetefrom a genus selected from the group consisting of Pythium,Phytophthora, and combinations thereof.

In some embodiments, said bacteria of the genus Bacillus compriseBacillus subtilis. In some embodiments, said bacteria of the genusBacillus comprise Bacillus subtilis var. amyloliquefaciens. In someembodiments, said bacteria of the genus Bacillus comprise Bacillussubtilis QST 713. In some embodiments, said bacteria of genus Collimonascomprise a species selected from the group consisting of Collimonasarenae, Collimonas fungivorans, and Collimonas pratensis. In someembodiments, said bacteria of genus Collimonas comprise C. arenae Cal35.In some embodiments, said bacteria of genus Collimonas comprise C.arenae Cal35 and said bacteria of the genus Bacillus comprise Bacillussubtilis QST 713.

In some embodiments, said bacteria of the genus Collimonas or a productthereof are in the form of a liquid suspension. In some embodiments,said bacteria of the genus Collimonas are in a liquid suspension at aconcentration of approximately 1×10⁶ cells per milliliter. In someembodiments, said bacteria of the genus Bacillus or a product thereofare in the form of a liquid suspension. In some embodiments, saidbacteria of the genus Bacillus are in a liquid suspension with aconcentration of approximately 1×10⁸ or 1×10⁹ colony forming units pergram.

In some embodiments, said contacting of a) orb) comprises a root dip. Insome embodiments, said contacting of a) or b) comprises a soil drench.In some embodiments, said contacting of a) and b) comprises a root dip.In some embodiments, said contacting of a) and b) comprises a soildrench. In some embodiments, said contacting of a) and b) areindependently selected from the group consisting of root dip, soildrench, and spray.

In a second aspect, the present invention provides a plant cultivated byany of the foregoing methods. In some embodiments, the present inventionprovides a plant in contact with bacteria from the genus Bacillus or aproduct thereof, and bacteria from the genus Collimonas, or a productthereof. In some embodiments, the plant is cultivated in soil that doesnot otherwise contain bacteria of the genus Collimonas.

In a third aspect, the present invention provides a composition ofmatter comprising: a) bacteria of the genus Collimonas or a productthereof; and b) bacteria of the genus Bacillus or a product thereof. Insome embodiments, the composition comprises bacteria of the genusCollimonas and a product thereof. In some embodiments, the compositioncomprises bacteria of the genus Bacillus and a product thereof. In someembodiments, the composition comprises bacteria of the genus Bacillusand bacteria of the genus Collimonas, wherein the bacteria of the genusCollimonas are not naturally found in agricultural soil.

In some embodiments, said bacteria of the genus Bacillus compriseBacillus subtilis. In some embodiments, said bacteria of the genusBacillus comprise Bacillus subtilis QST 713. In some embodiments, saidbacteria of genus Collimonas comprise a species selected from the groupconsisting of Collimonas arenae, Collimonas fungivorans, and Collimonaspratensis.

In some embodiments, said bacteria of the genus Collimonas are in theform of a liquid suspension. In some embodiments, said bacteria of thegenus Collimonas are in a liquid suspension with a concentration ofapproximately 1×10⁶ cells per milliliter. In some embodiments, saidbacteria of the genus Bacillus are in the form of a liquid suspension.In some embodiments, said bacteria of the genus Bacillus are in a liquidsuspension with a concentration of approximately 1×10⁸ or 1×10⁹ colonyforming units per gram.

In a fourth aspect, the present invention provides a plant in contactwith: a) bacteria of the genus Collimonas or a product thereof; and b)bacteria of the genus Bacillus or a product thereof. In someembodiments, the plant is a cultivated plant that is not naturally incontact with bacteria of the genus Collimonas.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4^(th) ed. 2007); Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor,N.Y. 1989); Raven et al. PLANT BIOLOGY (7^(th) ed. 2004). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

The term “plant” refers to a cultivated plant. The term “plant” includeswhole plants, shoot vegetative organs/structures (e.g. leaves, stems andtubers), roots, flowers and floral organs/structures (e.g. bracts,sepals, petals, stamens, carpels, anthers and ovules), seed (includingembryo, endosperm, and seed coat) and fruit (the mature ovary), planttissue (e.g. vascular tissue, ground tissue, and the like) and cells(e.g. guard cells, egg cells, trichomes and the like), and progeny ofsame. The class of plants that can be used in the method of theinvention is generally as broad as the class of higher and lower plantsamenable to cultivation, including angiosperms (monocotyledonous anddicotyledonous plants), gymnosperms, and ferns. It includes plants of avariety of ploidy levels, including aneuploid, polyploid, diploid,haploid and hemizygous. Specific embodiments of plants useful for themethods and compositions of the present invention include but are notlimited to commercial food crops, and energy crops.

The invention has use over broad range of plants, including species fromthe genera Anacardium, Arabidopsis, Arachis, Asparagus, Atropa, Avena,Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Oryza,Panieum, Pannesetum, Persea, Phaesolus, Pistachia, Pisum, Pyrus, Prunus,Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum,Theobromus, Trigonella, Triticum, Vicia Vitis, Vigna, and, Zea.

In some embodiments, the plant is selected from the group consisting ofrice, maize, wheat, soybeans, cotton, canola, turfgrass, alfalfa,poplar, eucalyptus, switchgrass, sorghum, millet, miscanthus, sugarcane,pine, barley, tobacco, hemp, poppy, bamboo, canola, rape, sunflower,willow, or Brachypodium. In some embodiments, the plant selected fromthe group consisting of tomato, potato, sweet potato, cassava, beets,ginger, horseradish, radish, ginseng, turnip, any root or tuber crop,pepper, eggplant, ground cherry, tomatillo, okra, other fruitingvegetables, cucumber cantaloupe, melon, muskmelon, squash, watermelonand other cucurbit plants. In some embodiments, the plant is anornamental plant. In some embodiments, the plant is a vegetable- orfruit-producing plant. In some embodiments, the plant is a monocot. Insome embodiments, the plant is a dicot.

The term “plant” can refer to any potential plant propagation material.Such plant propagation material can include any generative part of aplant such as seeds and vegetative plant material such as cuttings andtubers (e.g., potatoes), which can be used for the multiplication of theplant. This includes seeds, roots, fruits, tubers, bulbs, rhizomes,shoots, sprouts and other parts of plants, including seedlings and youngplants, which are to be transplanted after germination or afteremergence from soil or other planting media. These young plants may alsobe protected before transplantation by a total or partial treatment bycontacting with an anti-fungal or anti-oomycetous composition, such asany of the anti-fungal or anti-oomycetous compositions described herein.

The term “planting media” as used herein refers to any media that cansupport plant growth. The term includes soil, as well as media such asrock, wool, vermiculite, etc. The terms “soil” or “plant environment”for plants in the practice of the method of the present invention mean asupport for use in culture of a plant and especially a support in whichroots are to be grown. The terms are not limited in material quality,but include any material that may be used so far as a plant can be growntherein. For instance, so-called various soils, seedling mat, tapes,water or hydroponic solutions and the like can also be used. Specificexamples of the material constituting the soil or cultivation carrierinclude, without limitation, sand, peat moss, perlite, vermiculite,cotton, paper, diatomaceous earth, agar, gelatinous materials, polymericmaterials, rock wool, glass wool, wood chips, bark, pumice and the like.In some cases, the planting media is media that can support plant growthand does not naturally contain bacteria of the genus Collimonas.

As used herein, the term “fungus,” “fungal pathogen,” “fungi,” and thelike refers to a wide variety of nucleated organisms of the kingdomFungi that can infect or otherwise injure a plant. Examples of fungiinclude yeasts, molds, mildews, and rusts.

As used herein the term “plant resistant to a fungal or oomycetouspathogen” refers to a plant that is resistant to infection by a fungalor oomycetous pathogen or resistant to the symptoms of fungal oroomycetous pathogen infection. For example, a plant resistant to afungal or oomycetous pathogen can exhibit a lack of infection, orreduced symptoms of infection, when challenged with a pathogen. Asanother example, a plant resistant to a fungal or oomycetous pathogencan be infected by the fungal or oomycetous pathogen and yet exhibit areduced number or degree of symptoms of said infection. As yet anotherexample, a plant resistant to a fungal or oomycetous pathogen can beinfected by the pathogen and exhibit one or more symptoms of infectionby the pathogen and yet exhibit a reduction in an effect of theinfection or symptom thereof. For instance, a plant resistant to afungal or oomycetous pathogen can be infected by the pathogen, andexhibit one or more symptoms selected from the group consisting of leafwilt, leaf or vascular discoloration (e.g., yellowing), and yet exhibita reduction in yield loss in comparison to a plant that is not resistantto the fungal or oomycetous pathogen.

Accordingly, “enhanced resistance to a fungal or oomycetous pathogen”refers to a phenotype in which a plant has greater health, growth,multiplication, fertility, vigor, strength (e.g., lodging resistance),yield, or less severe symptoms of vascular discoloration during or aftera fungal or oomycetous infection than an organism that does not haveenhanced resistance to the pathogen. Where a plant is tested forresistance, a control plant could be a non-treated plant from the sameplant line. The enhancement can be an increase of 0.1%, 0.2%, 0.3%,0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%,17%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more inhealth, growth, multiplication, fertility, vigor, strength (e.g.,lodging resistance), or yield, as compared to a control plant. Theenhancement can be a decrease of 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 1%,1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%,35%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in vascular discoloration ascompard to a control plant.

As used herein, the term “contacting” in the context of contacting aplant with a bacteria, or a product thereof, refers to any suitablemethod for bringing a bacteria, or a product thereof, in contact withthe plant. Such methods include, but are not limited to, dipping,dripping, spraying, root dipping, soil drenching, dabbing, painting,coating, blotting, fumigating, irrigating, atomizing, or soil injection.The contacting can be applied to the plant directly. For example, aplant can be dipped or otherwise contacted with one or more bacteria orproducts thereof. The contacting can be performed prior to planting ofthe plant, after the planting of the plant, or during transfer of aplant from one planting media to another. Alternatively, the contactingcan be applied to the plant indirectly. For example, a soil or otherplanting media can be contacted with one or more bacteria or productsthereof, and the plant contacted with the soil or other planting media.

As used herein, the term “bacteria of the genus Collimonas” refers tobacteria from the Collimonas genus of bacteria in the familyOxalobacteraceae, order Burkholderiales. Collimonas can grow in slightlyacidic sandy dune soils. Collimonas are generally not found inagricultural soils. The bacteria are characterized in their ability tohydrolyze the fungal cell wall component chitin. Species of Collimonasinclude but are not limited to Collimonas arenae, Collimonasfungivorans, and Collimonas pratensis. Strains of Collimonas include butare not limited to Ter strains, such as C. fungivorans Ter331, Ter14, orTer6, C. pratensis Ter91, or C. arenae Ter10; or Cal strains, such as C.arenae Cal35, C. fungivorans Cal2, Cal1, or Cal39, or C. pratensisCal31; or Collimonas sp. D-25. The Collimonas can be provided as apaste, a powder, a suspension in liquid, a colony, a streak, a stab, orin any other suitable manner.

As used herein the term “bacteria of the genus Bacillus” refers tobacteria from the Bacillus genus of bacteria in the family Bacillaceae,order Bacillales. Species of Bacillus include but are not limited toBacillus subtilis. Strains of Bacillus include but are not limited toBacillus subtilis var. amyloliquefaciens and Bacillus subtilis QST 713.Additional species and strains of Bacillus are described herein. TheBacillus can be provided as a paste, a powder, a suspension in liquid, acolony, a streak, a stab, or in any other suitable manner.

As used herein, the term “product thereof” in reference to a bacteria ofthe genus Collimonas or Bacillus refers to one or more primary orsecondary metabolites of the bacteria. For example, the product thereofcan be an anti-fungal or anti-oomycetous agent produced by the bacteria.Such anti-fungal or anti-oomycetous agents include, but are not limitedto small molecule anti-fungal agents such as lipopeptides, cycliclipopeptides, bacilysin, chlorotetain, iturin A, fengycin, surfactins,and surfactin A. In some cases, such anti-fungal or anti-oomycetousagents can include hydrolases, such as proteases, or enzymes thathydrolyze chitin. Alternatively, the products thereof can provideenhanced plant vigor or growth, thus allowing the plant to mount astronger defense against a fungal or oomycetous pathogen. For example,the products thereof can include phytic acid, favorable for freeingphosphate from soil, thus facilitating plant absorption and leading toenhanced plant vigor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Representative photographs of confrontation plates showing thescale that was used to score mycelial growth of fungi/Oomycetes in thepresence of different bacterial strains. Shown here are 5 bacteria inconfrontation with Sclerotium cepivorum on WYA plates. The plate on thefar right is a control, with no bacteria inoculated across the center ofthe agar surface. Scores range on a scale of 1 (near-complete inhibitionof mycelial growth) to 5 (mycelial growth not at all affected).

FIG. 2. Representative photographs of tomato stems showing variousdegrees of vascular discoloration: 0, no discoloration; 1, discolorationof <5% of the stem cross section; 2, 5-20% discoloration; 3, 20-40%discoloration; 4, >40% discoloration.

FIG. 3. Dry weight (shoot) and vascular discoloration (0-4 scale) ofgreenhouse-grown, Fol-challenged tomato plants that were treated with amixture of Cal35 and Serenade Soil, Cal35 only, Serenade Soil only, orthat were untreated. Data points with a y-axis value of zero representthe control plants (no Fol). Panels A through D represent 4 independentexperiments, which were carried out in A) April/May 2012 (high/lowgreenhouse temperatures of 31/16° C.), B) June/July 2012 (33/17° C.), C)December 2012/January 2013 (29/15° C.). Data points for treatment withCal35 and Serenade soil in panels A, C, and D have a significantly lowervascular discoloration score than the other treatment data points.

FIG. 4. Dry weight and vascular discoloration (0-4 scale) offield-grown, harvest-ready Fol-challenged tomato plants that weretreated with a mixture of Cal35 and Serenade Soil, Cal35 only, SerenadeSoil only, or that were untreated. Green data points represent thecontrol plants (no Fol). Experiment was carried out in Summer 2014. Datapoints for treatment with Cal35 and Serenade soil have a significantlylower vascular discoloration score than the other treatment data points.

FIG. 5. Marketable yield (A) and yield loss due to sunburn (B), in tonsper acre, at time of harvest, from the field experiment described in thetext and in FIG. 2. Bars labeled with the same letter in either panel Aor panel B were not significantly different from each other.

DETAILED DESCRIPTION

Described herein are methods and compositions for biocontrol of fungalor oomycetous pathogens. In some embodiments, a composition can containa Collimonas bacteria or a product thereof. In some embodiments, thecomposition can contain a Bacillus bacteria or a product thereof. Insome embodiments, the composition can contain a Collimonas bacteria or aproduct thereof and a Bacillus bacteria or a product thereof. In somecases, such compositions can provide synergistic control of fungal oroomycetous pathogens when contacted with a cultivated plant. Suchcompositions generally do not contact a cultivated plant in naturebecause Collimonas are not naturally found in agricultural soils orother planting media. See, de Boer et al., IJSEM. 2004 May; 54(Pt3):857-64. In some cases, agricultural soils are soils that do notcontain detectable amounts of naturally occurring bacteria of the genusCollimonas.

I. Compositions

Compositions described herein contain one or more biocontrol agents. Thebiocontrol agents can include bacteria from the genus Bacillus, or oneor more products thereof. The biocontrol agents can include bacteriafrom the genus Collimonas, or one or more products thereof. Thebiocontrol agents can include bacteria from the genus Bacillus, or oneor more products thereof, and bacteria from the genus Collimonas, or oneor more products thereof.

For example, the biocontrol agent can contain Collimonas bacteria andone or more products of Bacillus. As another example, the biocontrolagent can contain Bacillus bacteria and one or more products ofCollimonas. As yet another example, the biocontrol agent can containCollimonas bacteria and one or more products thereof and one or moreproducts of Bacillus. As yet another example, the biocontrol agent cancontain bacteria from the genus Bacillus and one or more productsthereof, and bacteria from the genus Collimonas and one or more productsthereof.

For example, the composition can be a mixture of i) a pure orsubstantially pure Bacillus culture and ii) a pure or substantially pureCollimonas culture. In some cases, the cultures are separated from agrowth media (e.g., by centrifugation or filtering) and mixed in or on asterile liquid or solid medium. In some cases, the sterile liquid orsolid medium can be, or contain, spent media (e.g., sterilized spentmedia), or one or more products derived therefrom. In some cases, thecultures, including spent media are mixed together to obtain acomposition described herein. In some cases, a Bacillus culture isharvested and mixed into a Collimonas culture. In some cases, aCollimonas culture is harvested and mixed into a Bacillus culture.

Generally, the bacteria are pure or substantially pure. For example, thebacteria can be in a powder or liquid suspension that contains no, orsubstantially no, other living microorganisms. For instance, thebacteria can represent at least 90%, 95%, 99%, 99.9%, or more of theliving prokaryotic organisms in a powder, liquid suspension, or othersuitable carrier, medium, or agricultural formulation.

The one or more products of Bacillus or Collimonas can be obtained frombacteria or spent media. For example, the products can be obtained byculturing the bacteria with culture media (e.g. liquid or solid media)to obtain cultured bacteria and spent media. In some cases, thecomposition is, or contains, spent media, or one or more purifiedcomponents thereof. In some cases, the spent media is harvested byseparating the media from the bacteria. In some cases, bacterialproducts thereof are purified from the bacteria or the spent media. Forexample, bacteria can be lysed and one or more products thereof can bepurified. Alternatively, the spent media can be harvested and bacterialproducts can be purified from the spent media. In some cases, thecomposition does not contain spent media. In some cases, bacteria arecultured, spent media is harvested and optionally sterilized (e.g., byfiltration, addition of anti-biotic agents, or heat treatment, such aswith an autoclave), and then mixed with the bacteria to provide a liquidsuspension.

In some embodiments, products of one or more Bacillus or Collimonasbacteria can include metabolites produced by the microorganismsincluding, but not limited to, antibiotics, enzymes, siderophores orgrowth promoting agents. Such metabolites can include, for example,zwittermicin A, kanosamine, polyoxine, enzymes such as alpha-amylase,chitinases, and pectinases, phytohormones and precursors thereof, suchas auxins, gibberellin-like substances, cytokinin-like compounds,lipopeptides such as iturins, plipastatins, or surfactins, e.g.agrastatin A, bacillomycin D, bacilysin, difficidin, macrolactin,fengycin, bacilysin, plipastatin A, plipastatin B, and bacilaene. Insome cases, the metabolites are lipopeptides, such as those produced byBacillus pumilis (NRRL Accession No, B-30087) or Bacillus subtilis AQ713(NRRL Accession No. B-21661). In some cases, the metabolites are iturinA, surfactin A, plipastatin A, or agrastatin A.

In some cases, the composition contains spent media from Bacillus, orone or more purified components thereof. In some cases, the compositioncontains spent media from Collimonas, or one or more purified componentsthereof. In some cases, the composition contains spent media fromBacillus and Collimonas, or one or more purified components fromBacillus or Collimonas or from both Collimonas and Bacillus. In somecases, the composition contains spent media from Collimonas but does notcontain spent media from Bacillus. In some cases, the compositioncontains spent media from Bacillus, but does not contain spent mediafrom Collimonas.

In some cases, the composition comprises cultures (e.g., isolated purecultures) of one or more Bacillus and one or more Collimonas bacteria,or products thereof. In some cases, the composition comprises bacterialsuspensions in a whole broth culture or a metabolite containingsupernatant or purified metabolite obtained from whole broth culture ofone or more Bacillus or Collimonas.

The bacteria or products thereof can be in a liquid (e.g., a solution orsuspension) or solid form. Exemplary solid forms include but are notlimited to a powder or a paste. The bacteria can be in an otherwisesterile carrier or medium. The medium can be liquid (e.g., a solution orsuspension of bacteria) or solid (e.g., a powder or paste).

The chemical or physical properties of a liquid (e.g., solution orsuspension) containing bacteria and/or products thereof can be adjustedusing methods known in the art. For example, the liquid can be adjustedto increase the shelf life (e.g., increase stability of anti-fungalproperties over time) of the composition. In some cases, the pH orisotonic strength of the suspension can be adjusted by addition ofacids, bases, salts, or water to the suspension. As another example,stabilizing agents such as enzyme (e.g., protease) inhibitors orantibiotics can be added to the suspension. In some cases, the volume oramount of liquid can be adjusted to obtain a concentration of one ormore of Collimonas or Bacillus bacteria or products thereof. In somecases, the liquid further contains surfactants. In some cases, theliquid further contains plant nutrients or micronutrients, including butnot limited to nitrogen or phosphorous. In some cases, the liquid is pHadjusted before or after addition of bacteria or products thereof. Insome cases, the liquid contains one or more pH buffering agents.

For example, the bacteria can be in the form of colonies, a streak, or astab on an otherwise sterile medium, such as agar. As another example,the bacteria can be in the form of a powder mixed with a water solublemedium such as whey, maltodextrin, maltodextrin M100, etc. The powdercan include a moisture binding agent or dessicant to reduce clumping orincrease the ease of handling. Such moisture binding agents ordessicants include, but are not limited to silica, balith, or calciumchloride. As another example, the bacteria, or one or more productsthereof, can be mixed with planting media. For example, the bacteria orone or more products thereof can be mixed with sterile soil. In somecases, the solid medium further contains surfactants. In some cases, thesolid medium further contains plant nutrients or micronutrients,including but not limited to nitrogen or phosphorous. In some cases, thesolid medium is pH adjusted before or after addition of bacteria orproducts thereof. In some cases, the solid medium contains one or morepH buffering agents. In some cases, the volume or amount of solid mediacan be adjusted to obtain a concentration of one or more of Collimonasor Bacillus bacteria or products thereof.

Exemplary concentrations of Bacillus in a composition of the presentinvention can include, but are not limited to, 1×10³ colony formingunits/g (cfu/g), 1×10⁴ cfu/g, 1×10⁵ cfu/g, 1×10⁶ cfu/g, 1×10⁷ cfu/g,1×10⁸ cfu/g, 1×10⁹ cfu/g, 1×10¹⁰ cfu/g, 1×10¹¹ cfu/g or 1×10¹² cfu/g.Exemplary concentrations of Bacillus in a composition of the presentinvention can include, but are not limited to, 1×10³-1×10¹² cfu/g,1×10⁴-1×10¹¹ cfu/g, 1×10⁵-1×10¹⁰ cfu/g, 1×10⁶-1×10⁹ cfu/g, or1×10⁶-1×10⁸ cfu/g. Exemplary concentrations of Bacillus in a compositionof the present invention can include, but are not limited to, 1×10³cells/mL, 1×10⁴ cells/mL, 1×10⁵ cells/mL, 1×10⁶ cells/mL, 1×10⁷cells/mL, 1×10⁸ cells/mL, 1×10⁹ cells/mL, 1×10¹⁰ cells/mL, 1×10¹¹cells/mL or 1×10¹² cells/mL. Exemplary concentrations of Bacillus in acomposition of the present invention can include, but are not limitedto, 1×10³-1×10¹² cells/mL, 1×10⁴-1×10¹¹ cells/mL, 1×10⁵-1×10¹⁰ cells/mL,1×10⁶-1×10⁹ cells/mL, or 1×10⁶-1×10⁸ cells/mL.

Exemplary concentrations of Collimonas in a composition of the presentinvention can include, but are not limited to, 1×10³ colony formingunits/g (cfu/g), 1×10⁴ cfu/g, 1×10⁵ cfu/g, 1×10⁶ cfu/g, 1×10⁷ cfu/g,1×10⁸ cfu/g, 1×10⁹ cfu/g, 1×10¹⁰ cfu/g, 1×10¹¹ cfu/g or 1×10¹² cfu/g.Exemplary concentrations of Collimonas in a composition of the presentinvention can include, but are not limited to, 1×10³-1×10¹² cfu/g,1×10⁴-1×10¹¹ cfu/g, 1×10⁵-1×10¹⁰ cfu/g, 1×10⁶-1×10⁹ cfu/g, or1×10⁶-1×10⁸ cfu/g. Exemplary concentrations of Collimonas in acomposition of the present invention can include, but are not limitedto, 1×10³ cells/mL, 1×10⁴ cells/mL, 1×10⁵ cells/mL, 1×10⁶ cells/mL,1×10⁷ cells/mL, 1×10⁸ cells/mL, 1×10⁹ cells/mL, 1×10¹⁰ cells/mL, 1×10¹¹cells/mL or 1×10¹² cells/mL. Exemplary concentrations of Collimonas in acomposition of the present invention can include, but are not limitedto, 1×10³-1×10¹² cells/mL, 1×10⁴-1×10¹¹ cells/mL, 1×10⁵-1×10¹⁰ cells/mL,1×10⁶-1×10⁹ cells/mL, or 1×10⁶-1×10⁸ cells/mL.

Compositions of the present invention may include formulation inertsadded to compositions comprising cells, cell-free preparations ormetabolites to improve efficacy, stability, or usability and/or tofacilitate processing, packaging or end-use application. Suchformulation inerts and ingredients may include carriers, stabilizationagents, nutrients, or physical property modifying agents, which may beadded individually or in combination. In some embodiments, the carriersmay include liquid materials such as water, oil, and other organic orinorganic solvents and solid materials such as minerals, polymers, orpolymer complexes derived biologically or by chemical synthesis. In someembodiments, the carrier is a binder or adhesive that facilitatesadherence of the composition to a plant part, such as a seed or root.See, for example, Taylor, A. G., et al., “Concepts and Technologies ofSelected Seed Treatments”, Anna. ev. Phytopathol. 28: 321-339 (1990).The stabilization agents may include anti-caking agents, anti-oxidationagents, dessicants, protectants or preservatives. The nutrients mayinclude carbon, nitrogen, or phosphors sources such as sugars,polysaccharides, oil, proteins, amino acids, fatty acids or phosphates.The physical property modifiers may include bulking agents, wettingagents, thickeners, pH modifiers, rheology modifiers, dispersants,adjuvants, surfactants, antifreeze agents or colorants. In someembodiments, the composition comprising cells, cell-free preparations ormetabolites produced by fermentation can be used directly with orwithout water as the diluent without any other formulation preparation.In some embodiments, the formulation inerts are added afterconcentrating fermentation broth and during and/or after drying.

In some cases, the Bacillus are in an endospore form. In some cases, theBacillus are not in an endospore form. In some cases, the Bacillus arein a mixture of endospore and vegetative states. In some cases, at leastabout 1% of the Bacillus are in an endospore state and less than about99% are in a vegetative state. In some cases, at least about 99% of theBacillus are in a vegetative state and less than about 1% are in anendospore state. In some cases, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,12%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%,90%, 95%, 99%, or more of the Bacillus are in an endospore state. Insome cases, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 95%, 99%, or moreof the Bacillus are in a vegetative state.

Biocontrol agents can be applied in combination with syntheticchemicals, such as pesticides, nematicides, miticides, or fungicides. Insome cases, a biocontrol agent containing Collimonas or a productthereof and Bacillus or a product thereof can be mixed with one or moreother chemical and non-chemical additives, adjuvants or treatments,wherein such treatments include but are not limited to chemical andnon-chemical fungicides, insecticides, miticides, nematicides,fertilizers, nutrients, minerals, auxins, growth stimulants and thelike. In some cases, the biocontrol agent allows the use of a loweramount of synthetic chemical to obtain the same degree of pathogencontrol.

The present invention is directed to a composition comprising at leastone Bacillus bacteria selected from the group consisting of Bacilluschitinosporous AQ746 (NRRL Accession No. B-21618), Bacillus mycoidesAQ726 (NRRL Accession No. B-21664), Bacillus pumilus (NRRL Accession No.B-30087), Bacillus pumilus AQ717 (NRRL Accession No. B-21662), Bacillussp. AQ175 (ATCC Accession No. 55608), Bacillus sp. AQ177 (ATCC AccessionNo. 55609), Bacillus sp, AQ178 (ATCC Accession No. 53522), Bacillussubtilis AQ743 (NRRL Accession No. B-21665), Bacillus subtilis AQ713(NRRL Accession No. B-21661), Bacillus subtilis AQ153 (ATCC AccessionNo. 55614), Bacillus thuringiensis BD #32 (NRRL Accession No. B-21 30),Bacillus thuringiensis AQ52 (NRRL Accession No. B-21 619), Bacillusthuringiensis subspec. kurstaki BMP 123, Bacillus subtilis AQ30002 (NRRLAccession No. B-50421), and Bacillus subtilis AG 30004 (NRRL AccessionNo. B-50455) and/or a mutant or variant of one or more of these strains,and/or a metabolite produced by the respective strain that exhibitsactivity (e.g., synergistic activity) against a fungal or oomycetouspathogen when contacted with a plant simultaneously or sequentially witha composition containing a Collimonas bacteria or a product thereof. Insome cases, the Bacillus is a wild-type, mutant, or variant of one ormore of the foregoing strains, and/or a metabolite produced by therespective strain that exhibits activity (e.g., synergistic activity)against a fungal and an oomycetous pathogen when contacted with a plantsimultaneously or sequentially with a Collimonas bacteria or a productthereof. In some cases, the composition contains a mixture of one ormore of the foregoing Bacillus bacteria, a mutant of such bacteria, orone or more products thereof. In some cases, one or more Bacillusbacteria in the composition are recombinant bacteria. In some cases, thecomposition contains a product of a recombinant Bacillus bacteria.

In some cases, the Bacillus is, or is derived from, a commercialbiocontrol agent such as a Serenade product from AgraQuest, e.g.,Serenade ASO, Serenade Biofungicide, Serenade Garden Disease, SerenadeMax, or Serenade Soil. In some cases, the Bacillus is, or is derivedfrom, a commercial biocontrol agent such as EcoGuard Biofungicide orGreen Releaf from Novozymes Biologicals. In some cases, the Bacillus is,or is derived from, a commercial biocontrol agent such as Kodiak fromBayer Crop Science. In some cases, the Bacillus is, or is derived from,a commercial biocontrol agent such as Taegro from Novozymes. OtherBacillus based commercial biocontrol agents are known in the art, andthe Bacillus can be, or can be derived from, one or more such commercialBacillus based biocontrol agents.

Additional strains of Bacillus include, but are not limited to, thosedescribed in WO 2013/178664; WO 98/21966; WO 99/09820; WO 00/58442; WO99/10477; WO 98/21967; WO 99/09819; WO 98/50422; WO 98/21964; U.S. Pat.No. 5,645,831; WO 98/21965; and WO 2012/087980, each of which are herebyincorporated by reference in their entirety for all purposes. In somecases, the Bacillus is a wild-type, mutant, or variant of one or more ofthe foregoing strains, and/or a metabolite produced by the respectivestrain that exhibits activity (e.g., synergistic activity) against afungal or oomycetous pathogen when contacted with a plant simultaneouslyor sequentially with a Collimonas bacteria or a product thereof.

The present invention is directed to a composition comprising at leastone Collimonas bacteria selected from the group consisting of Collimonasarenae, Collimonas fungivorans, and Collimonas pratensis. In some cases,the Collimonas is a Ter strain. In some cases, the Collimonas is a Calstrain. In some cases, the Collimonas is a strain of C. arenae. In somecases, the C. arenae strain is Cal35. In some cases, the Collimonas is aCal31 strain. In some cases, the Collimonas is a Cal35 strain. In somecases, the Collimonas is a Cal39 strain. In some cases, the Collimonasis a Cal1 strain. In some cases, the Collimonas is a Cal2 strain. Insome cases, the Collimonas is a Ter6 strain. In some cases, theCollimonas is a Ter10 strain. In some cases, the Collimonas is a Ter14strain. In some cases, the Collimonas is a Ter331 strain. In some cases,the Collimonas is a D-25 strain. In some cases, the Collimonas is aCollimonas bacteria described in Leveau et al., Environ Microbiol. 2010February; 12(2):281-92; Hoppener-Ogawa et al., Environ Microbiol. 2009June; 11(6):1444-52; or Hoppener-Ogawa et al., ISME J. 2009 February;3(2):190-8. In some cases, the Collimonas is a wild-type, mutant, orvariant of one or more of the foregoing strains, and/or a metaboliteproduced by the respective strain that exhibits activity (e.g.,synergistic activity) against a fungal or oomycetous pathogen whencontacted with a plant simultaneously or sequentially with a Bacillusbacteria or a product thereof. In some cases, the Collimonas is awild-type, mutant, or variant of one or more of the foregoing strains,and/or a metabolite produced by the respective strain that exhibitsactivity (e.g., synergistic activity) against a fungal and an oomycetouspathogen when contacted with a plant simultaneously or sequentially witha Bacillus bacteria or a product thereof. In some cases, the compositioncontains a mixture of one or more of the foregoing Collimonas bacteria,a mutant of such bacteria, or one or more products thereof. In somecases, one or more Collimonas bacteria in the composition arerecombinant bacteria. In some cases, the composition contains a productof a recombinant Collimonas bacteria.

Compositions of the present invention can provide synergistic control offungal or oomycetous pathogens. As used herein, the term “synergistic”when used in the context of fungal pathogen control can refer to theanti-fungal or anti-oomycetous activity of a composition containing aBacillus bacteria or a product thereof and a Collimonas bacteria or aproduct thereof, wherein the composition is more effective (e.g.,statistically significantly more effective) against a fungal oroomycetous pathogen than one or more, or any, of the individualcomponents.

A “variant” is a strain having all the identifying characteristics ofthe parent organism as indicated in this text and can be identified ashaving a genome that hybridizes under conditions of high stringency tothe genome of the parent organism.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. Hybridization reactions can be performed underconditions of different “stringency.” In general, a tow stringencyhybridization reaction is carried out at about 40° C. in 10×SSC or asolution of equivalent ionic strength/temperature. A moderate stringencyhybridization is typically performed at about 50° C. in 6×SSC, and ahigh stringency hybridization reaction is generally performed at about60° C. in 1×SSC.

A variant of an indicated microorganism (e.g., a microorganism indicatedby an NRRL or ATCC Accession Number, or a strain designation) may alsobe defined as a strain having a genomic sequence that is greater than85%, more preferably greater than 90% or more preferably greater than95% sequence identity to the genome of the indicated organism. Apolynucleotide or polynucleotide region (or a polypeptide or polypeptideregion) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of“sequence identity” to another sequence means that, when aligned, thatpercentage of bases (or amino acids) are the same in comparing the twosequences. This alignment and the percent homology or sequence identitycan be determined using software programs known in the art, for example,those described in Current Protocols in Molecular Biology (F. M, Ausubelet al. eds., 1987) Supplement 30, section 7. 7. 18, Table 7. 7. 1.

Compositions of the present invention can be used for control of a widevariety of fungal or oomycetous pathogens. The fungal or oomycetouspathogens can be one or more fungi or Oomycetes from a class selectedfrom the group consisting of Plasmodiophoromycetes, Oomycetes,Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes,Deuteromycetes, and Sordariomycetes. The fungal or oomycetous pathogenscan be one or more fungi from a genus selected from the group consistingof Fusarium, Geotrichum, Aspergillus, Alternaria, Botryosphaeria,Colletotrichum, Magnaporthe, Verticillium, Cryphonectria, Botrytis,Monilinia, Sclerotium, and Rhizoctonia. The fungal or oomycetouspathogen can be from the class Oomycetes (synonym Peronosporomycetes).In some cases, the fungal pathogen is Fusarium oxysporum. In some cases,the fungal pathogen is Fusarium oxysporum f. sp. lycopersici (Fol) race3. In some cases, the Fusarium oxysporum f sp. lycopersici (Fol) race 3is strain D11 or D12, or a variant thereof.

II. Formulations

Described herein are compositions as described above and additionallycomprising at least one additional component selected from the groupconsisting of extenders, solvents, spontaneity promoters, carriers,emulsifiers, dispersants, frost protectants, thickeners, and adjuvants.These compositions are referred to as formulations.

Described herein are formulations for application of one or morecompositions of the present invention to a plant or planting media. Inone aspect, such formulations, and application forms prepared from them,are provided as crop protection agents and/or fungicidal agents, such asdrench, drip and spray liquors, comprising a composition of theinvention. The application forms may comprise further crop protectionagents and/or pesticidal agents, and/or activity-enhancing adjuvantssuch as penetrants. For example, such application forms can comprisevegetable oils such as, for example, rapeseed oil, sunflower oil,mineral oils such as, for example, liquid paraffins, alkyl esters ofvegetable fatty acids, such as rapeseed oil or soybean oil methylesters, or alkanol alkoxylates, and/or spreaders such as, for example,alkylsiloxanes and/or salts, examples being organic or inorganicammonium or phosphonium salts, examples being ammonium sulphate ordiamonium hydrogen phosphate, and/or retention promoters such as dioctylsulphosuccinate or hydroxypropylguar polymers and/or humectants such asglycerol and/or fertilizers such as ammonium, potassium or phosphorousfertilizers, for example.

Examples of typical formulations include water-soluble liquids (SL),emulsifiable concentrates (EC), emulsions in water (EW), suspensionconcentrates (SC, SE, FS, OD), water-dispersible granules (WG), granules(G) and capsule concentrates (CS); these and other possible types offormulation are described, for example, by Crop Life International andin Pesticide Specifications, Manual on development and use of FAO andWHO specifications for pesticides, FAO Plant Production and ProtectionPapers—1 73, prepared by the FAO/WHQ Joint Meeting on PesticideSpecifications, 2004, ISBN: 9251 048576. The formulations may compriseactive agrochemical compounds other than one or more active componentsof the invention (e.g., other than Collimonas or a product thereof andBacillus or a product thereof).

The formulations or application forms in question preferably compriseauxiliaries, such as extenders, solvents, spontaneity promoters,carriers, emulsifiers, dispersants, frost protectants, biocides,thickeners and/or other auxiliaries, such as adjuvants, for example. Anadjuvant in this context is a component which enhances the biologicaleffect of the formulation, without the component itself having abiological effect. Examples of adjuvants are agents which promote theretention, spreading, attachment to the plant or penetration of theplant surface (e.g., penetration of the root, shoot, or leaf surface).

Suitable extenders are, for example, water, polar and nonpolar organicchemical liquids, for example from the classes of the aromatic andnon-aromatic hydrocarbons (such as paraffins, alkylbenzenes,alkylnaphthalenes, or chlorobenzenes), the alcohols and polyols (which,if appropriate, may also be substituted, etherified and/or esterified),the ketones (such as acetone, cyclohexanone), esters (including fats andoils) and (poly)ethers, the unsubstituted and substituted amines,amides, lactams (such as N-alkylpyrrolidones) and lactones, thesulphones and sulphoxides (such as dimethyl sulphoxide).

If the extender used is water, it is also possible to employ, forexample, organic solvents as auxiliary solvents. Suitable liquidsolvents include, but are not limited to: aromatics such as xylene,toluene or alkylnaphthalenes, chlorinated aromatics or chlorinatedaliphatic hydrocarbons such as chlorobenzenes, chloroethylenes ormethylene chloride, aliphatic hydrocarbons such as cyclohexane orparaffins, for example petroleum fractions, mineral or vegetable oils,alcohols such as butanol or glycol and also their ethers and esters,ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone orcyclohexanone, strongly polar solvents such as dimethyl formamide ordimethyl sulphoxide, or water.

Any suitable carrier may in principle be used. Suitable carriers caninclude, for example, ammonium salts and ground natural minerals such askaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite, ordiatomaceous earth, or ground synthetic minerals, such as finely dividedsilica, alumina or natural or synthetic silicates, resins, waxes and/orsolid fertilizers. Mixtures of such carriers may likewise be used.Carriers suitable for granules include the following; for example,crushed or fractionated natural minerals such as calcite, marble,pumice, sepiolite, dolomite, or synthetic granules of inorganic andorganic meals, or granules of organic material such as sawdust, paper,coconut shells, maize cobs or tobacco stalks.

Examples of emulsifiers and/or foam-formers, dispersants or wettingagents having ionic or nonionic properties, or mixtures of thesesurface-active substances, are salts of polyacrylic acid, salts oflignosulphonic acid, salts of phenolsulphonic acid ornaphthalenesulphonic acid, polycondensates of ethylene oxide with fattyalcohols or with fatty acids or with fatty amines, with substitutedphenols (preferably alkylphenois or arylphenols), salts ofsulphosuccinic esters, taurine derivatives (preferably alkyltaurates),phosphoric esters of polyethoxylated alcohols or phenols, fatty acidesters of polyols, or derivatives of the compounds containing sulphates,sulphonates, phosphates, or phosphonates, examples being alkylarylpolyglycol ethers, alkylsulphonates, alkyl sulphates, arylsulphonates,protein hydrolysates, lignin-sulphite waste liquors and methylcellulose.

Further auxiliaries that may be present in the formulations and in theapplication forms derived from them include colorants such as inorganicpigments, examples being iron oxide, titanium oxide, Prussian Blue, andorganic dyes, such as alizarin dyes, azo dyes or metal phthalocyaninedyes, or nutrients or trace nutrients, such as salts of iron, manganese,boron, copper, cobalt, molybdenum or zinc.

Stabilizers, such as low-temperature stabilizers, preservatives,antioxidants, light stabilizers or other agents which improve chemical,biological, and/or physical stability may also be present. Additionallypresent may be foam-formers or defoamers.

Furthermore, the formulations and application forms derived from them,may also comprise, as additional auxiliaries, adherents such ascarboxymethylcellulose, natural and synthetic polymers in powder,granule or latex form, such as gum arable, polyvinyl alcohol, polyvinylacetate, or natural phospholipids, such as cephalins or lecithins, orsynthetic phospholipids. Further possible auxiliaries include mineral orvegetable oils.

There may possibly be further auxiliaries present in the formulationsand the application forms derived from them. Examples of such additivesinclude fragrances, protective colloids, binders, adhesives, thickeners,thixotropic substances, penetrants, retention promoters, stabilizers,sequestrants, complexing agents, humectants or spreaders.

Suitable retention promoters include any of those substances whichreduce the dynamic surface tension, such as dioctyl sulphosuccinate, orincrease viscoelasticity, such as hydroxypropylguar polymers, forexample.

Suitable penetrants in the present context include all those substanceswhich are typically used in order to enhance the penetration of activeagrochemical compounds into plants. Penetrants in this context aredefined in that, from a (generally aqueous) application liquor and/orfrom a spray coating, they are able to penetrate the cuticle of theplant and thereby increase the mobility of one or more active compoundsin the cuticle. This property can be determined using the methoddescribed in the literature (Baur et al., 1997, Pesticide Science 51,131-152). Examples include alcohol alkoxylates such as coconut fattyethoxylate or isotridecyl ethoxylate, fatty acid esters such as rapeseedor soybean oil methyl esters, fatty amine alkoxylates such astaliowamine ethoxylate, or ammonium and/or phosphonium salts such asammonium sulphate or diammonium hydrogen phosphate, for example.

The formulations can comprise between 0.00000001% and 98% or 99% byweight of active component or, between 0.01% and 95% by weight of activecomponent, or between 0.5% and 90% by weight of active component, basedon the weight of the formulation. The content of the active component isdefined as the sum of Bacillus bacteria or product thereof andCollimonas bacteria or product thereof.

The active component content of the application forms (crop protectionproducts) prepared from the formulations may vary within wide ranges.The active compound concentration of the application forms may besituated typically between 0.00000001% and 95% by weight of activecomponent, or between 0.00001% and 1% by weight, based on the weight ofthe application form. Application can be performed in a customary manneradapted to the application forms.

Generally speaking, the active components may be combined with any solidor liquid additive commonly used for formulation purposes. Typically,the formulation additives are selected from those additives that do notinactivate the active components. For example, additives that do notinactivate Bacillus or Collimonas present in a composition, or productsthereof, may be used in the formulations described herein.

III. Methods

Compositions or formulations as described above can be applied to aplant to reduce overall damage of plants and plant parts as well aslosses in harvested fruits or vegetables caused by fungal or oomycetousphytopathogens. In some cases, a composition or formulation describedabove can be applied to a plant to increase resistance to a fungal oroomycetous pathogen. In some cases, the composition as described abovecan be applied to a plant to increase the overall plant health.

The term “plant health” generally refers various aspects of plant growthor resistance to external insults. For example, a plant with increasedplant health may exhibit one or more of the following improved cropcharacteristics including: emergence, crop yields, protein content, oilcontent, starch content, more developed root system, improved rootgrowth, improved root size maintenance, improved root effectiveness,improved stress tolerance (e.g., against drought, heat, salt, UV, water,cold), reduced ethylene (reduced production and/or inhibition ofreception), tillering increase, increase in plant height, bigger leafblade, less dead basal leaves, stronger tillers, greener leaf color,pigment content, photosynthetic activity, less input needed (such asfertilizers or water), less seeds needed, more productive tillers,earlier flowering, early grain maturity, less plant verse (e.g., lesslodging), increased shoot growth, enhanced plant vigor, increased plantstand and early and better germination.

The effect of a composition according to the present invention on planthealth as defined herein can be determined by comparing plants which aregrown under the same environmental conditions, whereby a part of saidplants is treated with a composition according to the present inventionand another part of said plants is not treated with a compositionaccording to the present invention. In some cases, said other part isnot treated at all, treated with a placebo (e.g., an application withouta composition according to the invention such as an application withonly Bacillus or only Collimonas). In some cases, said other part istreated with a conventional treatment with a known efficacy.

A composition according to the present invention can be applied in anydesired manner, such as in the form of a seed coating, soil drench,and/or directly in-furrow and/or as a foliar spray and applied eitherpre-emergence, post-emergence or both. The composition can be applied tothe seed, the plant or to harvested fruits and vegetables or to theplanting media (e.g., soil) wherein the plant is growing or wherein itis desired to grow. In some cases, the composition is applied during,before, or shortly after, transfer of the plant from one planting mediato another. For example, a plant may be grown in a greenhouse until itreaches a certain stage, harvested, treated, and transferred to a field.A composition described herein can be applied to a conventional ortransgenic plant.

In some embodiments, a composition containing Bacillus or a productthereof can be contacted with a plant and simultaneously or sequentiallya composition containing a Collimonas can be contacted with the plant.The contacting can be performed by a wide variety of methods known inthe art.

In some cases, the plant is contacted with a formulation containing aCollimonas or product thereof and a Bacillus or product thereofformulated into a single, stable composition with an agriculturallyacceptable shelf life. In some cases, one or more compositions orcomponents are combined before or at the time of use. In some cases, aformulation is in a single “ready-mix” form. In some cases, theformulation is a combined spray mixture composed from solo-formulationsthat are combined during the application process, such as in a “tankmix” formulation. In some case, one or more components of thecomposition are combined when they are contacted with a plant in asequential manner, i.e., one after the other within a reasonably shortperiod, such as a few hours or days, e.g., 0.5, 1, or 2 hours to 7 days.The order of applying the composition according to the present inventionis not essential for working the present invention.

Contacting a composition or formulation containing a compositiondescribed herein to a plant can be performed using a wide variety ofcustomary treatment methods. For example the plant can contacted bydipping, coating, seed coating, spraying, atomizing, irrigating,evaporating, dusting, fogging, broadcasting, foaming, painting,spreading-on, watering (drenching), soil injection, or drip irrigating.

The amount of composition or formulation that is contacted with theplant depends on the final formulation, the size or type of the plant,plant parts, seeds, harvested fruits and vegetables to be treated, andthe nature of the fungal or oomycetous pathogen, or degree of infection.In one embodiment a composition is contacted with a plant, thecomposition containing a Bacillus bacteria in a concentration of atleast 10⁵ colony forming units per gram preparation (e.g., cells/gpreparation, spores/g preparation), such as 10⁵-10¹² cfu/g, 10⁶-10¹¹cfu/g, 10⁷-10¹⁰ cfu/g, or 10⁹-10¹⁰ cfu/g at the time point of applyingbiological control agents on a plant or plant parts such as seeds,fruits or vegetables. In one embodiment a composition is contacted witha plant, the composition containing a Collimonas bacteria in aconcentration of at least 10⁵ colony forming units per gram preparation(e.g., cells/g preparation, spores/g preparation), such as 10⁵-10¹²cfu/g, 10⁶-10¹¹ cfu/g, 10⁷-10¹⁰ cfu/g, or 10⁹-10¹⁰ cfu/g at the timepoint of applying biological control agents on a plant or plant partssuch as seeds, fruits or vegetables.

The Collimonas or a product thereof and Bacillus or a product thereof,can be used or employed in a synergistic ratio (e.g., by weight, mass,colony forming units, or cells per unit volume). The synergistic ratioscan be determined routine methods. These ratios can refer to the ratiowithin a combined-formulation as well as to the ratio when bothcomponents are applied as mono-formulations to a plant to be treated.

In one embodiment the synergistic ratio of Bacillus bacteria toCollimonas bacteria lies in the range of 1:1000 to 1000:1, in the rangeof 1:500 to 500:1, in the range of 1:100 to 100:1, in the range of 1:10to 10:1, in the range of 1:2 to 2:1, or about 1:1. In this context suchratios refer to a ratio of the number of cells or colony forming unitsof Bacillus over the number of cells or colony forming units ofCollimonas bacteria contacted to a plant.

In one embodiment of the present invention, the concentration ofCollimonas or Bacillus after dispersal is at least 50 g/ha, such as50-7500 g/ha, 50-2500 g/ha, 50-1500 g/ha; at least 250 g/ha, at least500 g/ha or at least 800 g/ha. The application rate of composition to beemployed or used according to the present invention may vary. Theskilled person is able to find the appropriate application rate by wayof routine experiments.

In some embodiments, one or more compositions of the present inventionare contacted with a plant by applying the composition to a seed. Insome cases, the seed is contacted with Bacillus or a product thereof. Insome cases, the seed is contacted with Collimonas or a product thereof.In some cases, the seed is contacted with Bacillus or a product thereofand Collimonas or a product thereof. In some cases, the seed is treatedat different times with Bacillus or a product thereof and thenCollimonas or a product thereof. In some cases, the seed is treated atdifferent times with Collimonas or a product thereof and then Bacillusor a product thereof. In some cases, one or more individualcompositions, or components thereof, of the invention may be present indifferent layers on the seed. In some cases, following treatment withthe composition of the invention, the seed is subjected to afilm-coating process in order to prevent dust abrasion of the seed.

A composition of the invention can be applied alone or in a suitableformulation to the seed. The seed can be treated in a condition suchthat no damage occurs in the course of the treatment. Generallyspeaking, the seed may be treated at any point in time betweenharvesting and sowing. Typically, seed is used which has been separatedfrom the plant and has had cobs, hulls, stems, husks, hair or pulpremoved. Thus, for example, seed may be used that has been harvested,cleaned and dried to a moisture content of less than 15% by weight.Alternatively, seed can also be used that after drying has been treatedwith water, for example, and then dried again.

When treating seed it is necessary, generally speaking, to ensure thatthe amount of the composition of the invention, and/or of otheradditives, that is applied to the seed is selected such that thegermination of the seed is not adversely affected, and/or that the plantwhich emerges from the seed is not damaged. This is the case inparticular with active ingredients which may exhibit phytotoxic effectsat certain application rates.

The compositions of the invention can be applied in the form of asuitable formulation to the seed. Suitable formulations and methods forseed treatment are known to the skilled person and are described in, forexample, the following documents; U.S. Pat. No. 4,272,417; U.S. Pat. No.4,245,432; U.S. Pat. No. 4,808,430; U.S. Pat. No. 5,876,739; US2003/0176428; WO 2002/080675; or WO 2002/0281 86, the contents of whichare hereby incorporated in their entirety for all purposes.

The application rate of the seed-dressing formulations which can be usedin accordance with the invention may be varied within a relatively widerange. It is guided by the particular amount of the Bacillus or productsthereof or by the particular amount of Collimonas or products thereof,by the nature of the fungal or oomycetous pathogen, by the seed, orcombinations thereof.

Compositions of the present invention can be contacted with a plant toprotect the plant from phytopathogenic fungi or Oomycetes. Suchphytopathogenic fungi or Oomycetes include, but are not limited to,soilborne pathogens, which include members of the classesPlasmodiophoromycetes, Peronosporomycetes, (Syn, Oomycetes),Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes andDeuteromycetes (Syn. Fungi imperfecti). The compositions can be used inplant protection as foliar, seed dressing or soil fungicide.Furthermore, they are suitable for combating fungi or Oomycetes, whichinter alia infest wood or roots of plant.

Non-limiting examples of pathogens of fungal diseases which can betreated in accordance with the invention include: diseases caused bypowdery mildew pathogens, for example Blumeria species, for exampleBlumeria graminis; Podosphaera species, for example Podosphaeraleucotricha; Sphaerotheca species, for example Sphaerotheca fuliginea;or Uncinula species, for example Uncinula necator.

One or more compositions of the invention can be contacted with a plantto control diseases caused by rust disease pathogens, for exampleGymnosporangium species, for example Gymnosporangium sabinae; Hemileiaspecies, for example Hemileia vastatrix; Phakopsora species, for examplePhakopsora pachyrhizi and Phakopsora meibomiae; Puccinia species, forexample Puccinia recondite, P. triticina, P. graminis, or P.striiformus; or Uromyces species, for example Uromyces appendiculatus.

One or more compositions of the invention can be contacted with a plantto control diseases caused by pathogens from the group of the Oomycetes,for example Albugo species, for example Algubo Candida; Bremia species,for example Bremia lactucae; Peronospora species, for examplePeronospora pisi or P. brassicae; Phytophthora species, for examplePhytophthora infestans; Plasmopara species, for example Plasmoparaviticola; Pseudoperonospora species, for example Pseudoperonosporahumuli or Pseudoperonospora cubensis; or Pythium species, for examplePythlum ultimum.

One or more compositions of the invention can be contacted with a plantto control leaf blotch diseases and leaf wilt diseases caused, forexample, by Alternaria species, for example Alternaria solani;Cercospora species, for example Cercospora beticola; Cladiosporiumspecies, for example Cladiosporium cucumerinum; Cochliobolus species,for example Cochliobolus sativus, Cochliobolus miyabeanus;Colletotrichum species, for example Colletotrichum lindemuthianum;Cycloconium species, for example Cycloconium oleaginum; Diaporthespecies, for example Diaporthe citri; Elsinoë species, for exampleElsinoë fawcettii; Gloeosporium species, for example Gloeosporiumlaeticolor; Glomerella species, for example Glomerella cingulata;Gulgnardia species, for example Gulgnardia bidwelli; Leptosphaeriaspecies, for example Leptosphaeria maculans, Leptosphaeria nodorum;Magnaporthe species, for example Magnaporthe grisea; Microdochiumspecies, for example Microdochium nivale; Mycosphaerella species, forexample Mycosphaerella graminicola, M. arachidicola and M. fijiensis;Phaeosphaeria species, for example Phaeosphaeria nodorum; Pyrenophoraspecies, for example Pyrenophora teres; Ramularia species, for exampleRamularia collo-cygni; Rhynchosporium species, for exampleRhynchosporium secalis; Septoria species, for example Septoria apii;Typhula species, for example Typhula incarnate; or Venturia species, forexample Venturia inaequalis.

One or more compositions of the invention can be contacted with a plantto control root and stem diseases caused, for example, by Corticiumspecies, for example Corticium graminearum; Fusarium species, forexample Fusarium oxysporum; Gaeumannomyces species, for exampleGaeumannomyces graminis; Rhizoctonia species, such as, for exampleRhizoctonia solani; Tapesia species, for example Tapesia acuformis; orThielaviopsis species, for example Thielaviopsis basicola.

One or more compositions of the invention can be contacted with a plantto control ear and panicle diseases (including corn cobs) caused, forexample, by Alternaria species, for example Alternaria spp.; Aspergillusspecies, for example Aspergillus flavus; Cladosporium species, forexample Cladosporium cladosporioides; Claviceps species, for exampleClaviceps purpurea; Fusarium species, for example Fusarium culmorum;Gibberella species, for example Gibberella zeae; Monographella species,for example Monographella nivalis; or Septoria species, for exampleSeptoria nodorum.

One or more compositions of the invention can be contacted with a plantto control diseases caused by smut fungi, for example Sphacelothecaspecies, for example Sphacelotheca reiliana; Tilletia species, forexample Tilletia caries, T. controversa; Urocystis species, for exampleUrocystis occulta; or Ustilago species, for example Ustilago nuda, or U.nuda tritici.

One or more compositions of the invention can be contacted with a plantto control fruit rot caused, for example, by Aspergillus species, forexample Aspergillus flavus; Botrytis species, for example Botrytiscinerea; Penicillium species, for example Penicillium expansum and P.purpurogenum; Sclerotinia species, for example Sclerotinia sclerotiorum;or Verticilium species, for example Verticilium alboatram.

One or more compositions of the invention can be contacted with a plantto control seed- and soil-borne rot and wilt diseases, and also diseasesof seedlings, caused, for example, by Fusarium species, for exampleFusarium culmorum; Phytophthora species, for example Phytophthoracactorum; Pythium species, for example Pythium ultimum; Rhizoctoniaspecies, for example Rhizoctonia solani; or Sclerotium species, forexample Sclerotium rolfsii.

One or more compositions of the invention can be contacted with a plantto control cancers, galls and witches' broom caused, for example, byNectria species, for example Nectria galligena; or wilt diseases caused,for example, by Monilinia species, for example Monilinia laxa.

One or more compositions of the invention can be contacted with a plantto control deformations of leaves, flowers and fruits caused, forexample, by Taphrina species, for example Taphrina deformans.

One or more compositions of the invention can be contacted with a plantto control degenerative diseases of woody plants caused, for example, byEsca species, for example Phaeomoniella chlamydospora, Phaeoacremoniumaleophilum, or Fomitiporia mediterranea.

One or more compositions of the invention can be contacted with a plantto control diseases of flowers and seeds caused, for example, byBotrytis species, for example Botrytis cinerea; diseases of plant tuberscaused, for example, by Rhizoctonia species, for example Rhizoctoniasolani; or Helminthosporium species, for example Helminthosporiumsolani.

The following diseases can be controlled using one or more compositionsof the present invention: Fungal diseases on leaves, stems, pods andseeds caused, for example, by Alternaria leaf spot (Alternaria spec.atrans tenuissima), Anthracnose (Colletotrichum gloeosporoides dematiumvar. truncatum), brown spot (Septoria glycines), cercospora leaf spotand blight (Cercospora kikuchii), Choanephora leaf blight (Choanephorainfundibulifera trispora (Syn.)), dactuliophora leaf spot (Dactuliophoraglycines), downy mildew (Peronospora manshurica), drechslera blight(Drechslera glycini), frogeye leaf spot (Cercospora sojina),leptosphaerulina leaf spot (Leptosphaerulina trifolii), phyllostica leafspot (Phyllosticta sojaecola), pod and stem blight (Phomopsis sojae),powdery mildew (Microsphaera diffusa), pyrenochaeta leaf spot(Pyrenochaeta glycines), rhizoctonia aerial, foliage, and web blight(Rhizoctonia solani), rust (Phakopsora pachyrhizi, Phakopsorameibomiae), scab (Sphaceloma glycines), stemphylium leaf blight(Stemphylium botryosum), target spot (Corynespora cassiicola).

Fungal diseases on roots and the stem base caused, for example, by blackroot rot (Calonectria crotalariae), charcoal rot (Macrophominaphaseolina), fusarium blight or wilt, root rot, and pod and collar rot(Fusarium oxysporum, Fusarium orthoceras, Fusarium semitectum, Fusariumequiseti), mycoleptodiscus root rot (Mycoleptodiscus terrestris),neocosmospora (Neocosmospora vasinfecta), pod and stem blight (Diaporthephaseolorum), stem canker (Diaporthe phaseolorum var. caulivora),phytophthora rot (Phytophthora megasperma), brown stem rot (Phialophoragregata), pythium rot (Pythium aphanidermatum, Pythium irregulare,Pythium debaryanum, Pythium myriotylum, Pythium ultimum), rhizoctoniaroot rot, stem decay, and damping-off (Rhizoctonia solani), sclerotiniastem decay (Sclerotinia sclerotiorum), sclerotinia southern blight(Sclerotinia rolfsii), thielaviopsis root rot (Thielaviopsis basicola).

Plants which can be treated in accordance with the invention include thefollowing main crop plants: maize, soya bean, cotton, Brassica oil seedssuch as Brassica napus (e.g. canola), Brassica rapa, B. juncea (e.g.(field) mustard) and Brassica carinata, rice, wheat, sugar beet, sugarcane, oats, rye, barley, millet and sorghum, triticale, flax, grapes andvarious fruit and vegetables from various botanic taxa, for exampleRosaceae sp. (for example pome fruits such as apples and pears, but alsostone fruits such as apricots, cherries, almonds and peaches, and berryfruits such as strawberries), Ribesioidae sp., Juglandaceae sp.,Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceaesp., Actimidaceae sp., Lauraceae sp., Musaceae sp. (for example bananatrees and plantations), Rubiaceae sp. (for example coffee), Theaceaesp., Sterculiceae sp., Rutaceae sp. (for example lemons, oranges andgrapefruit); Solanaceae sp. (for example tomatoes, potatoes, peppers,aubergines), Liliaceae sp., Compositae sp. (for example lettuce,artichokes and chicory—including root chicory, endive or commonchicory), Umbelliferae sp. (for example carrots, parsley, celery andceleriac), Cucurbitaceae sp. (for example cucumbers—including gherkins,pumpkins, watermelons, calabashes and melons), Alliaceae sp. (forexample leeks and onions), Cruciferae sp. (for example white cabbage,red cabbage, broccoli, cauliflower, Brussels sprouts, pak choi,kohlrabi, radishes, horseradish, cress and chinese cabbage), Leguminosaesp. (for example peanuts, peas, and beans—for example common beans andbroad beans), Chenopodiaceae sp. (for example Swiss chard, fodder beet,spinach, beetroot), Malvaceae (for example okra), Asparagaceae (forexample asparagus); useful plants and ornamental plants in the gardenand woods; and in each case genetically modified types, varietals, orcultivars of these plants.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Antifungal and Antioomycete Properties of Collimonas Isoaltesand their Use in Synergy-Based Biocontrol of Fusarium wilt

I. Introduction

The bacterial genus Collimonas (Leveau, J. H., S. Uroz, et al. (2010)Environmental Microbiology 12(2): 281-292) comprises three describedspecies: C. fungivorans, C. pratensis, and C. arenae (de Boer, W., J. H.J. Leveau, et al. (2004) International Journal of Systematic andEvolutionary Microbiology 54: 857-864; Höppener-Ogawa, S., W. de Boer,et al. (2008) International Journal of Systematic and EvolutionaryMicrobiology 58: 414-419). Collimonads have antifungal (de Boer, W., P.J. A. Klein Gunnewiek, et al. (1998) Soil Biology & Biochemistry 30(2):193-203; Mela, F., K. Fritsche, et al. (2011) ISME J5(9): 1494-1504),mycophagous (de Boer, W., P. J. A. Klein Gunnewiek, et al. (2001)Applied and Environmental Microbiology 67(8): 3358-3362), chitinolytic(Leveau, J. H. J., S. Gerards, et al. (2006) Journal of MicrobiologicalMethods 66(2): 276-285), and weathering (Uroz, S., C. Calvaruso, et al.(2009) Soil Biology & Biochemistry 41(10): 2178-2186) properties. C.fungivorans Ter331 is the best characterized Collimonas strain so far:its genome sequence is available (Mela, F., K. Fritsche, et al. (2008)FEMS Microbiology Ecology 66(1): 45-62; Mela, F., K. Fritsche, et al.(2011) ISME J 5(9): 1494-1504; Mela, F., K. Fritsche, et al. (2012)Environmental Microbiology Reports 4(4): 424-432), its chitinolyticsystem has been elucidated (Fritsche, K., W. de Boer, et al. (2008) FEMSMicrobiology Ecology 66(1): 123-135), and it was shown to berhizosphere-competent and to protect tomato plants from developingsymptoms upon infection with Fusarium oxysporum f. sp.radicis-lycopersici which causes Fusarium crown and root rot on tomato(Kamilova, F., J. H. J. Leveau, et al. (2007) Environmental Microbiology9(6): 1597-1603). Strain Ter331 was one of several so-called Ter strainsthat were isolated from dune soils on the Dutch island of Terschelling(de Boer, W., P. J. A. Klein Gunnewiek, et al. (1998) Soil Biology &Biochemistry 30(2): 193-203). Since then, the presence of collimonadshas been reported from many other parts of the world (Offre, P., B.Pivato, et al. (2008) FEMS Microbiology Ecology 65(2): 180-192; Postma,J., M. T. Schilder, et al. (2008) Soil Biology & Biochemistry 40(9):2394-2406; Axelrood, P. E., M. L. Chow, et al. (2002) Canadian Journalof Microbiology 48(7): 655-674; Chow, M. L., C. C. Radomski, et al.(2002) FEMS Microbiology Ecology 42(3): 347-357. Project Collifornia waslaunched in 2009 to discover Collimonas strains native to California.Here, we provide a characterization of these Cal strains in terms ofantifungal and antioomycete activity in vitro. We show that strain C.arenae Cal35 was by far the most effective in these confrontationassays. We also demonstrate that in a greenhouse setting a mixture of C.arenae Cal35 and the commercially available, Bacillus-based biofungicideSerenade Soil, but not Cal35 alone or Serenade Soil alone, can reducedisease symptoms on tomato plants that were challenged with Fusariumoxysporum f. sp. lycopersicum (Fol), causative agent of Fusarium wilt.We refer to this synergy-based emerging property as ‘biocombicontrol’.

II. Materials and Methods

Microorganisms used in this study. Collimonas strains Ter6, Ter10,Ter14, Ter91 and Ter331 have been described elsewhere (de Boer, W., P.J. A. Klein Gunnewiek, et al. (1998) Soil Biology & Biochemistry 30(2):193-203; de Boer, W., J. H. J. Leveau, et al. (2004) InternationalJournal of Systematic and Evolutionary Microbiology 54: 857-864;Hoppener-Ogawa, S., W. de Boer, et al. (2008) International Journal ofSystematic and Evolutionary Microbiology 58: 414-419). Collimonasstrains Cal1 and Cal2 were isolated from soil near Berry Creek, Calif.,while Cal31, Cal35, and Cal39 were isolated from forest soil at theJughandle Reserve, Mendocino County, Calif. (Uroz, S., J. J. Tech, etal. (2013) Biology & Biochemistry, in press). The methods for isolatingand identifying Collimonas isolates have been described in detailelsewhere (Uroz, S., J. J. Tech, et al. (2013) Biology & Biochemistry,in press). In short, soil samples were inoculated into Tryptic Soy Brothcontaining natamycin and nalidixic acid, incubated at 20° C. for 3 dayswhile shaking, after which an aliquot was transferred to chitin brothfor an additional incubation at 20° C. for 7 days. In parallel, soilsamples were seeded directly into chitin broth. Aliquots of chitin brothenrichments were spread onto colloidal chitin agar plates, and incubatedat 20° C. for 7 days. Colonies with a halo of cleared chitin wereselected and analyzed using a Collimonas-specific probe assay(Hoppener-Ogawa et al., Appl Environ Microbiol. 2007 July;73(13):4191-7). Positive isolates were further tested by a RestrictionFragment Length Polymorphism (RFLP) assay on amplified 16S rRNA genesusing the BstBI restriction enzyme, which is specific for theidentification of Collimonas (Hoppener-Ogawa et al., Appl EnvironMicrobiol. 2007 July; 73(13):4191-7). Positive isolates were confirmedto be Collimonas and assigned to one of the three described species byDNA sequencing of PCR-amplified 16S rRNA genes using primers pA and1492r. An additional Collimonas strain, Collimonas sp. D-25, was a kindgift from Yoichiro Hirose and was originally isolated from forestrhizosphere soil in Japan. Fungi and Oomycetes were gifts from variousfaculty members in the Department of Plant Pathology at UC Davis. Manyof them cause disease on economically important crops and trees. For thegreenhouse experiments (see below), two different strains of Fusariumoxysporum f sp. lycopersici (Fol) race 3 were used. Strain D11 wasoriginally isolated from infected tomato plants on a commercial field inYolo County, Calif., and had been in storage on sterile filter papersince 2010. Strain D12 was isolated in 2012 from infected plants in SanJoaquin County, Calif. Isolation of Fol was achieved by washing stems ofdiseased plants with anti-bacterial hand soap (Dial Gold), and cuttingdiagonally to produce 0.25-cm thick pieces which were surface sterilizedin 0.6% sodium hypochloride (10% bleach) for 1 minute, and placed onAcidified Potato Dextrose Agar (APDA) plates for 5 days at 25° C. withan 8-hour photoperiod (48 W fluorescence light bulbs, Lights of America,model 8045E, Los Angeles, Calif.). After 5 days, hyphal tips of coloniespresenting Fusarium-like macroconidia were transferred to fresh APDAplates and incubated for another 7 days. From the resulting colonies,single spores were transferred to individual APDA plates. DNA wasextracted from 30 mg of hyphal mass with the DNeasy Plant Mini Kit(Qiagen, Valencia, Calif.) and used in a PCR with Fusarium-specificprimers EF-1 (5′-ATGGGTAAGGAAGACAAGAC-3′) and EF-2(5′-GGAAGTACCAGTGATCATGTT-3′). The PCR was conducted in final volumes of50 μl per reaction, using 10 ng of template DNA, 0.2 mM of each dNTP,0.2 μM of each primer, and 0.25 units of GoTaq DNA polymerase. Thefollowing PCR cycling conditions were used: 40 cycles of 30 sec. at 95°C., 30 sec. at 55° C., and 1 min at 72° C., then followed by 5 min. at72° C. The PCR products were visualized on 1.5% TBE agarose gels. Testsof pathogenicity and race differentiation were performed with tomatocultivars Early Pak 7 (susceptible), VFN-8 (resistant to race 1), Walter(resistant to race 1 and 2), and AB 319 (resistant to race 1, 2, and 3).

Confrontation assays. To test the antagonistic activity of collimonads(n=11) and control bacteria (n=2) against our set of fungi and Oomycetes(n=22), we used a confrontation setup that has been described previouslyby Mela, F., K. Fritsche, et al. (2011) ISME J 5(9): 1494-1504. Thisinvolved streaking the bacteria against spores or a plug of fungus oroomycete on Water Yeast Agar (WYA) supplemented with 2 mMN-acetylglucosamine. To score the antagonistic activity of eachbacterial strain against every one of the fungi/Oomycetes, we used ascale from 1 to 5, where 1 represents strong inhibition and 5 representscomplete lack of inhibition (FIG. 1). In total, we looked at 286pairwise confrontations (13 bacterial strains×22 fungal/oomycetalstrains). Each confrontation assay was performed in triplicate, and amean score was calculated for each pair. To calculate anantifungal/antioomycete' score for any given bacterial strain, weaveraged the mean scores over all 22 fungi/Oomycetes tested against thatstrain. To calculate a ‘Collimonas-susceptibility’ score for any givenfungus/oomycete, we averaged the mean scores over all 11 Collimonasstrains tested against that fungus/oomycete.

Greenhouse experiments. In total, we performed 4 independent greenhouseexperiments to test if and by how much C. arenae Cal35, Serenade Soil,or a mixture of Cal35 and Serenade Soil could reduce the weight loss andvascular discoloration that result from infection of tomato seedlingswith Fol D11 or D12. Fungal spore suspensions were prepared fresh fromone-week-old APDA cultures of Fol. The colonized plates were floodedwith 15 mL of autoclaved deionized water, then conidia were scraped offusing a sterile glass slide and filtered through four layers of sterilecheesecloth. The conidial concentration was determined with a NeubauerLevy Ultra Plane Hemacytometer (Hausser Scientific, Horsham, Pa.) andadjusted to 10⁶ per ml with autoclaved deionized water. C. arenae Cal35was streaked from −80° C. freezer stock onto King's B agar and incubatedat 24° C. for 5 days. Plates were then flooded with 15 mL of autoclaveddeionized water and bacterial biomass was scraped off with a sterileglass slide. Serial dilutions were made of this suspension, read foroptical density at 600 nm (OD₆₀₀) using a Genesys 10S UV-Visspectrophotometer (Thermo Scientific, Wilmington, Del.), and adjusted to10⁶ cells per ml using autoclaved deionized water. Suspensions ofSerenade Soil (Agraquest, Davis Calif.) were prepared by 1000-folddilution of the liquid product (containing 10⁹ colony-forming units ofBacillus per gram, as per the label) in water. Mixtures of Cal35 andSerenade Soil (referred to as Collinade) were prepared just prior to usein the experiments by mixing equal volumes of Serenade Soil suspensionand C. arenae Cal 35 suspension.

Tomato seeds (Early Pak 7) were sown in the greenhouse in containerswith UC potting soil mix (50% sphagnum peat moss, 50% washed sand, andper cubic meter-2.43 kg of dolomite lime, 0.872 kg oyster shell lime,0.872 kg superphosphate, 0.344 kg calcium nitrate, 135 ml potassiumnitrate, and 90 ml potassium sulfate). After 11 days, seedlings (twotrue leaves) were gently removed from the soil, root-dipped for 4minutes in water, Cal35 suspension, Serenade Soil suspension, orCollinade, then transferred to fresh UC potting soil mix, and placedback in the greenhouse. After one week, seedling roots were dug up againand dipped for 4 minutes in either water or FOL conidial suspension,then transplanted into containers with fresh UC potting soil mix. After3 days, 100 ml of water or Cal35, Serenade Soil, or Collinade werepoured onto the soil surrounding the crown of the tomato plant. One weeklater, this drench was repeated. Four weeks after the root inoculationwith Fol, individual tomato plants were cut at the soil line todetermine shoot dry weight and to assess vascular discoloration in thestem on a scale of 0-4 (see FIG. 2: 0, no vascular discoloration; 1, <5%discoloration, typically light brown; 2, 5-20% discoloration, typicallylight brown; 3, 20-40% discoloration, typically dark brown; 4, >40%discoloration, typically dark brown). Data were analyzed using SASversion 5.1.2600 (SAS Institute, Inc. Cary N.C.). Plants were grown inthe greenhouse in a randomized complete block design, with 6 replicatesper treatment in experiment 1, 10 replicates in experiments 2 and 3, and15 replicates in experiment 4.

Field trial. We designed and carried out a field trial during the 2014growing season at the Armstrong Plant Pathology Field Station at UCDavis. Tomato plants (Heinz 5508, resistant to Fol race 1 and 2 but notrace 3, also resistant to Verticillium dahliae race 1 and Tomato SpottedWilt Virus) were seeded in 128-cell seedling trays with 3.5×3.5×6.5 cmcells and kept in the greenhouse for three weeks, at which time trayswere dipped for 4 minutes in one of the following four solutions(prepared as described above): 1) water, 2) Cal35 suspension, 3)Serenade Soil suspension, or 4) Collinade. Dipped trays were placed backin the greenhouse and after one week, tomato plants werehand-transplanted (May 17, 2014) 30 cm apart into a drip-irrigatableplot with a randomized complete block design (five single-row 92-cm bedswith 60-cm between-row spacing beds, plus a pair of border rows)featuring 20 blocks with 30 plants each: 8 of these blocks were plantedwith water-dipped seedlings, 4 with Cal35-dipped seedlings, 4 withSerenade Soil-dipped seedlings, and 4 with Collinade-dipped seedlings.Immediately after planting, 10 liters of Fol spore suspension weredelivered by drip to all blocks except for 4 of the blocks planted withthe water-dipped seedlings; these blocks represent the no-Fol treatment.The spore solution was prepared as described above. After one week, 10liters of water, Cal35 suspension, Serenade Soil suspension, orCollinade were drip-delivered into each one of the corresponding blocks(e.g., Cal35 suspension was delivered into the blocks that were plantedwith Cal35-dipped seedlings, and water into the blocks with water-dippedseedlings). One week later, this injection was repeated. The field waswatered as needed through the drip. Fertilizer UN-32 was applied throughthe drip once a week until flowering, at 75 ml per block and starting 12days after transplanting. Plants also received a one-time application ofMiracle-Gro Quick Start (Scotts Miracle-Gro, Marysville, Ohio) 5 daysafter transplanting, at 450 ml per block. Weeding was done manually, noherbicides or pesticides were used. Fruits were harvested on Sep. 19,2014 (4 months after planting), at which time more than 90 percent ofthe fruit were ripe. Vascular discoloration and shoot dry weight (afterremoval of the fruit, see below) were determined for 15 plants in thecenter of each block, as described above. Also measured for these 15plants combined were the weight of red, green, sunburn, and moldy fruit.Marketable yield was calculated from red fruit weight, and expressed intons per acre.

III. Results

We tested 11 strains of Collimonas (Table 1) in standardizedconfrontation assays (Mela, F., K. Fritsche, et al. (2011) ISME J 5(9):1494-1504) to assess and compare their impact on mycelial growth of 22fungi or Oomycetes (Table 2). Collimonas strains belonged to one of thethree known species (C. arenae, fungivorans or pratensis), and included5 of the original Ter isolates, in addition to 5 isolates recovered fromCalifornian soils and a Japanese strain Collimonas sp. D-25. We alsoincluded two negative controls, i.e. E. coli TOP10 (Invitrogen) and P.putida 1290 (Leveau, J. H. J., and S. E. Lindow. (2005) Applied andEnvironmental Microbiology, 71(5): 2365-2371; Leveau, J. H. J. and S.Gerards. (2008) FEMS Microbiology Ecology 65(2): 238-250). In total, 286bacterium-fungus/oomycete pairs were evaluated in triplicate on WaterYeast Agar (WYA) plates supplemented with 2 mM N-acetylglucosamine asdescribed before (Mela, F., K. Fritsche, et al. (2011) ISME J5(9):1494-1504) and scored on a scale from 1 (near-complete inhibition ofmycelial growth) to 5 (complete lack of inhibition of mycelial growth)as shown in FIG. 1.

TABLE 1 Bacterial strains used in this study and tested for theirability to inhibit mycelial growth. Genus Species Strain^(a) Score^(a)Collimonas arenae Cal35 1.75 ± 0.89 Collimonas fungivorans Ter14 2.06 ±0.99 Collimonas fungivorans Ter6 2.30 ± 1.01 Collimonas fungivorans Cal22.30 ± 1.21 Collimonas fungivorans Cal1 2.37 ± 1.12 Collimonasfungivorans Cal39 2.47 ± 1.11 Collimonas pratensis Ter91 2.75 ± 1.12Collimonas arenae Ter10 2.84 ± 1.00 Collimonas sp. D-25 2.90 ± 1.59Collimonas fungivorans Ter331 2.91 ± 1.10 Collimonas pratensis Cal313.04 ± 1.10 Pseudomonas putida 1290 5.02 ± 0.24 Escherichia coli TOP105.06 ± 0.27 ^(a)Shown is the average and standard deviation of all meanscores (each mean score calculated from 3 replicate assays perfungus/oomycete) for each bacterial strain in confrontation with 22tested fungi/Oomycetes. Scoring was done as shown in FIG. 1: 1 meansnear-complete inhibition while 5 means no inhibition at all. Strains areranked according to their score.

TABLE 2 Fungi and Oomycetes used in this study and their relativesusceptibility to growth inhibition by Collimonas strains. Phylum ClassOrder Family Genus Species Score^(a) Ascomycota SaccharomycetesSaccharomycetales Dipodascaceae Geotrichum candidum ¹ 1.05 ± 0.17Eurotiomycetes Eurotiales Trichocomaceae Aspergillus niger ² 1.42 ± 0.55carbonarius ³ 2.50 ± 1.06 Dothideomycetes Pleosporales PleosporaceaeAlternaria alternata ⁴ 2.77 ± 0.66 Botryosphaeriales BotryosphaeriaceaeBotryosphaeria stevensii ⁵ 2.36 ± 1.11 Sordariomycetes Hypocreales —Fusarium circinatum ⁶ 3.91 ± 0.30 oxysporum ⁷ 3.27 ± 0.65 GlomerellaceaeColletotrichum acutatum ⁸ 2.84 ± 0.81 Magnaporthales MagnaporthaceaeMagnaporthe grisea ⁹ 2.76 ± 0.73 Phyllachorales — Verticillium dahliae¹⁰ 2.33 ± 0.92 Diaphorthales Cryphonectriaceae Cryphonectria parasitica¹¹ 1.91 ± 0.79 Leotiomycetes Helotiales Sclerotiniaceae Botrytis cinerea¹² 1.04 ± 0.13 Monilinia fructicola ¹³ 1.00 ± 0.00 BasidiomycotaAgaricomycetes Atheliales Atheliaceae Sclerotium rolfsii ¹⁴ 2.27 ± 0.91cepivorum ¹⁵ 1.64 ± 1.12 Cantharellales Ceratobasidiaceae Rhizoctoniasolani ¹⁶ 3.09 ± 0.92 Heterokontophyta Oomycetes Pythiales PythiaceaePythium violae ¹⁷ 3.14 ± 1.28 irregulare ¹⁸ 2.36 ± 1.13 ultimum ¹⁹ 3.17± 1.16 Peronosporales Pythiaceae Phytophthora capsici ²⁰ 3.09 ± 0.94cactorum ²¹ 4.00 ± 0.00 Zygomycota Zygomycetes Mucorales MucoraceaeMucor sp.²² 3.46 ± 0.69 ^(a)Shown is the average and standard deviationof all mean scores (each mean score calculated from 3 replicate assaysper Collimonas strain) for each fungal/oomycetal strain in confrontationwith 11 tested collimonads. Scoring was done as shown in FIG. 1: 1 meansnear-complete inhibition while 5 means no inhibition at all. ¹causativeagent of sour rot on fruits and vegetables; ²model strain CBS120.49;³sour rot of grape; ⁴tomato mold; ⁵tree cankers; ⁶pitch canker of pine;⁷f. sp. lycopersici, race 3, tomato wilt; ⁸fruit rot; ⁹rice blast; ¹⁰Verticillium wilt of tomato; ¹¹chestnut blight; ¹²bunch rot of grape;¹³brown rot of peach; ¹⁴Southern blight; ¹⁵ Allium root rot; ¹⁶dampingoff disease; ¹⁷cavity spot of carrot; ^(18/19)damping off disease ofcarrot; ²⁰rot of bell pepper; ²¹root rots; ²²common soil fungus.

Averaged over all tested fungi/Oomycetes (Table 1), the inhibitoryperformance was greatest for strain C. arenae Cal 35 (score=1.75),followed by C. fungivorans Ter14 (2.06) and C. fungivorans Cal 2 (2.30).Strain Cal35 was unique and superior among the tested Collimonas strainsin that it was the only one to near-completely inhibit the growth ofSclerotium rolfsii and Rhizoctonia solani as well as the OomycetesPythium violae, Pythium ultimum, and Phytophthora capsici. It sharedwith C. fungivorans Ter14 the ability to inhibit Pythium irregulare.Strain Cal35 was also the sole best inhibitor of Fusarium circinatum,Fusarium oxysporum, and Mucor sp. The overall least affected byco-inoculation with Collimonas bacteria (Table 2) were the fungusFusarium circinatum (score=3.9) and the oomycete Phytophthora cactorum(4.0). Three fungi were near-completely inhibited (<1.7) by allCollimonas strains tested, i.e. Monilinia fructicola, Botrytis cinerea,and Geotrichum candidum. All three are known as important postharvestpathogens of fruits, including tomato. None of the fungi tested werenegatively affected by E. coli or P. putida (>4.50). In some cases,fungal growth actually seemed to be stimulated by the presence of E.coli or P. putida (e.g. as was the case for Phytophthora capsici).

C. arenae strain Cal35 was selected for further experiments to test thehypothesis that its in vitro observed antifungal activity predicts itspotential as a biocontrol agent to protect plant roots against fungalpathogens. In a first of 4 greenhouse experiments (FIG. 3A), whenapplied as a prophylactic root dip and subsequent soil drenches, Cal 35was not able to suppress the effects of Fusarium wilt (measured asvascular discoloration and dry weight) on tomato plants that wereroot-dip inoculated with Fol race 3 isolate D11 (FIG. 3A, first andthird circles from the top). In a control treatment with the commercial,Bacillus-based product Serenade Soil, we saw the same result (FIG. 3A,second circle from the top). However, when Cal35 was applied as a 1:1mixture with Serenade Soil, we observed a significant reduction invascular discoloration and a decrease in dry weight loss (FIG. 3A,fourth circle from the top). In two independent repetitions of thisexperiment with another Fol isolate (D12), we achieved the same resultin one of the experiments (FIG. 1D); in the other (FIG. 3B), theCal35-Serenade Soil mixture had no effect. During the latter experiment,greenhouse temperatures were higher than in any of the other greenhousetrials; possibly, this was the reason for biocontrol failure in thiscase. A fourth greenhouse experiment featured both D11 and D12, andagain we observed protection of the tomato seedlings from Fol with theCal35/Serenade Soil mixture, but not Cal35 alone or Serenade Soil alone(FIG. 3C).

In a controlled trial at the Plant Pathology Field Station at UC Daviswhich was designed to replicate the greenhouse experiments in a fieldsetting, we observed the same synergistically Collinade protection fromFol-induced symptoms, i.e. vascular discoloration and dry weight loss(FIG. 4). Vascular discoloration in the Collinade treatment wassignificantly reduced compared to Cal35 alone (p=0.00024) or Serenade(p=0.00067) alone. Similarly, the Collinade treatment yielded asignificantly higher shoot dry weight (after removal of the fruit) thanCal35 alone (p=0.0032) or Serenade alone (p=0.0084). Marketable fruityield was highest with Collinade (68.3 tons per acre), but this valuedid not differ significantly from other treatments, including the no-Folcontrol, at 56.3 tons per acre (FIG. 5A). However, we did observe (FIG.5B) significantly less (approximately 1 ton per acre) of sunburn-relatedloss of yield in Fol-challenged plots that received Collinade, comparedto Fol-challenged plots that received Serenade alone (p=0.046) or notreatment (p=0.043).

IV. Conclusion

This study demonstrates and compares the antifungal and antioomyceteactivities of Collimonas strains isolated from Californian soils. The invitro data suggest not only variation in the ability of individualCollimonas strains to inhibit or slow down the mycelial growth of fungiand/or Oomycetes, but also in the relative susceptibility offungal/oomycetal strains to confrontation with Collimonas. In greenhouseand field experiments, we were able to demonstrate a synergistic effectbetween C. arenae Cal35 and Soil Serenade which protected tomato plantsfrom Fol symptoms including vascular discoloration and dry weight loss.In the field experiment, we saw no statistically significant impact ofFol on marketable yield, but we did observe a protective effect fromsunburn damage. We suspect that this is related to our observation (notshown) of a thicker canopy in the Collinade-treated blocks which wouldbetter protect the developing fruit from sun exposure. A thicker canopyalso explains the higher shoot dry weight, which in turn follows fromthe ability of Collinade to prevent vascular infection by Fol. While themechanisms underlying the observed synergism between Cal35 and SerenadeSoil are unknown still, the data seem to suggest that there is enormouspotential for improvement of the efficacy of already existing productssuch as Serenade Soil through combination with bacteria such asCollimonas to achieve biocombicontrol of plant diseases.

Example 2 Soil Sample Analysis of Collimonads

A total of 24 soil samples were analyzed for the presence ofcollimonads. These samples were taken as triplicates from 8 agriculturalfields in California that were planted with tomatoes, almonds, alfalfa,strawberry, wheat, lettuce, onion or carrot. Analysis was performedusing an enrichment protocol as described before. In short, one gramfrom each soil sample was resuspended in 10 mL of 1× Basic Salt Solution(de Boer, W., Klein Gunnewiek, P. J. A., Lafeber, P., Janse, J. D.,Spit, B. E., Woldendorp, J. W., 1998, Anti-fungal properties ofchitinolytic dune soil bacteria. Soil Biology and Biochemistry.30,193-203) by vortexing at maximum speed for 45 seconds. One milliliterof each suspension was inoculated into 50 ml of tryptic soy broth(Oxoid) with 50 mg natacid and 30 mg nalidixic acid per liter andincubated at 20° C. for 3 days at 200 rpm, after which a 100-μl aliquotwas transferred to 50 mL of chitin broth (modified from de Boer, W.,Leveau, J. H. J., Kowalchuk, G. A., Klein Gunnewiek, P. J. A., Abeln, E.C. A., Figge, M. J., Sjollema, K., Janse, J. D., van Veen, J. A., 2004,Collimonas fungivorans gen. nov., sp. nov., a chitinolytic soilbacterium with the ability to grow on living fungal hyphae.International Journal of Systematic and Evolution Microbiology 54,857-864, excluding the agar) supplemented with 50 mg natacid and 30 mgnalidixic acid per liter for an additional incubation at 20° C. for 7days at 200 rpm. In parallel, one milliliter of the original soilsuspension was inoculated directly into 50 ml of chitin brothsupplemented with 50 mg natacid and 30 mg nalidixic acid per liter andincubated at 20° C. for 7 days at 200 rpm. Ten microliters of the chitinbroth enrichments were spread in duplicate onto chitin agar plates (deBoer, W., Leveau, J. H. J., Kowalchuk, G. A., Klein Gunnewiek, P. J. A.,Abeln, E. C. A., Figge, M. J., Sjollema, K., Janse, J. D., van Veen, J.A., 2004, Collimonas fungivorans gen. nov., sp. nov., a chitinolyticsoil bacterium with the ability to grow on living fungal hyphae.International Journal of Systematic and Evolution Microbiology 54,857-864), and incubated at 20° C. for 7 days. Colonies with a halo ofcleared chitin were shown to be negative in a Collimonas-specific probeassay (Hoppener-Ogawa, S., Leveau, J. H., Smant, W., van Veen, J. A., deBoer, W., 2007. Specific detection and real-time PCR quantification ofpotentially mycophagous bacteria belonging to the genus Collimonas indifferent soil ecosystems. Applied and Environmental Microbiology. 73,4191-4197). This result was confirmed by a Restriction Fragment LengthPolymorphism (RFLP) assay on amplified 16S rRNA genes using the BstBIrestriction enzyme, which is specific for the identification ofCollimonas (Hoppener-Ogawa, S., Leveau, J. H., Smant, W., van Veen, J.A., de Boer, W., 2007. Specific detection and real-time PCRquantification of potentially mycophagous bacteria belonging to thegenus Collimonas in different soil ecosystems. Applied and EnvironmentalMicrobiology. 73, 4191-4197) and by DNA sequencing of PCR amplified 16SrRNA genes using primers pA (Edwards, U., Rogall, T., Blocker, H., Emde,M., Bottger, E. C. 1989. Isolation and direct complete nucleotidedetermination of entire genes. Characterisation of a gene coding for 16Sribosomal RNA. Nucleic Acids Research. 17, 7843-7853) and 1492r (Lane,D. J., 1991. 16S/23S rRNA sequencing, p. 115-176. In E. Stackebrandt M.Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. JohnWiley and Sons, Chichester, United Kingdom). In conclusion, bacteriafrom the genus Collimonas in these soil samples were undetectable by themethods employed.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-39. (canceled)
 40. A method of cultivating a plant comprising: a)contacting the plant with Bacillus subtilis var. amyloliquefaciensstrain FZB24, or a product thereof; and b) contacting the plant withbacteria of the genus Collimonas, or a product thereof, wherein saidcontacting of a) in combination with said contacting of b) causes asynergistic antifungal or synergistic anti-oomycete effect against afungal or an oomycetous infection.
 41. The method of claim 40, whereinthe part of said plant contacted with bacteria of the genus Collimonasor a product thereof is a root of said plant.
 42. The method of claim40, wherein the part of said plant contacted with Bacillus subtilis var.amyloliquefaciens strain FZB24 or a product thereof is a root of saidplant.
 43. The method of claim 40, wherein the part of said plantcontacted with bacteria of the genus Collimonas or a product thereof isa seed of said plant.
 44. The method of claim 40, wherein the part ofsaid plant contacted with Bacillus subtilis var. amyloliquefaciensstrain FZB24 or a product thereof is a seed of said plant.
 45. Themethod of claim 40, wherein the part of said plant contacted withbacteria of the genus Collimonas or a product thereof is foliage of saidplant.
 46. The method of claim 40, wherein the part of said plantcontacted with Bacillus subtilis var. amyloliquefaciens strain FZB24 ora product thereof is foliage of said plant.
 47. The method of claim 40,wherein the contacting of b) comprises contacting the plant withbacteria of the genus Collimonas and a product thereof.
 48. The methodof claim 40, wherein the contacting of a) comprises contacting the plantwith Bacillus subtilis var. amyloliquefaciens strain FZB24 and a productthereof.
 49. The method of claim 40, wherein the plant is a cucurbitplant.
 50. The method of claim 40, wherein said bacteria of genusCollimonas comprise a species selected from the group consisting ofCollimonas fungivorans, and Collimonas pratensis.
 51. The method ofclaim 40, wherein said bacteria of genus Collimonas comprise Collimonasarenae.
 52. The method of claim 40, wherein said bacteria of genusCollimonas comprise C. arenae Cal35.
 53. The method of claim 40, whereinsaid bacteria of the genus Collimonas or a product thereof are in theform of a liquid suspension.
 54. The method of claim 40, wherein saidbacteria of the genus Collimonas are in a liquid suspension at aconcentration of from about 1×10⁴ to about 1×10¹¹ cells per milliliter.55. The method of claim 40, wherein said Bacillus subtilis var.amyloliquefaciens strain FZB24 is in a liquid suspension at aconcentration of from about 1×10⁴ to about 1×10¹¹ cells per milliliter.56. The method of claim 40, wherein the contacting of a) or b) comprisesa root dip.
 57. The method of claim 40, wherein the contacting of a) orb) comprises a soil drench.
 58. The method of claim 40, wherein theplant is a cucurbit.
 59. A plant in contact with: a) Bacillus subtilisvar. amyloliquefaciens strain FZB24; and b) a bacteria of the genusCollimonas, wherein a) and b) together exhibit a synergistic antifungalor synergistic anti-oomycetous effect.
 60. The plant of claim 59,wherein the plant is a cucurbit.